A high-throughput assay for the comprehensive profiling of DNA ligase fidelity.
A high-throughput assay for the comprehensive profiling of DNA ligase fidelity.
复制标题
DOI：
10.1093/nar/gkv898
复制
发表时间：
2016-01-29
影响因子：
14.9
通讯作者：
Evans TC Jr
中科院分区：
文献类型：
--
作者：
Lohman GJ;Bauer RJ;Nichols NM;Mazzola L;Bybee J;Rivizzigno D;Cantin E;Evans TC Jr
DNA连接酶在分子生物学中具有广泛的应用，从传统的克隆方法到现代合成生物学和分子诊断方案。多核苷酸序列的基于连接的检测可以通过探针寡核苷酸与互补靶序列退火时的连接来实现。为了实现高灵敏度和低背景，连接酶必须有效地连接正确的碱基配对的底物，同时区分含有甚至一个错配碱基对的底物的连接。在目前的研究中，我们报告了使用毛细管电泳快速生成的错配保真度配置文件，询问所有256个可能的碱基对组合在一个单一的实验连接交界处。在96孔板格式中快速筛选连接酶保真度使得对连接酶保真度的研究达到前所未有的深度。作为这种新方法的一个例子，本文中，我们报告了嗜热栖热菌DNA连接酶在一系列温度，缓冲液pH值和单价阳离子强度下的连接保真度。该筛选允许选择在不牺牲活性的情况下使保真度最大化的反应条件，同时产生在每组条件下可检测地连接的特异性错配的概况。
DNA ligases have broad application in molecular biology, from traditional cloning methods to modern synthetic biology and molecular diagnostics protocols. Ligation-based detection of polynucleotide sequences can be achieved by the ligation of probe oligonucleotides when annealed to a complementary target sequence. In order to achieve a high sensitivity and low background, the ligase must efficiently join correctly base-paired substrates, while discriminating against the ligation of substrates containing even one mismatched base pair. In the current study, we report the use of capillary electrophoresis to rapidly generate mismatch fidelity profiles that interrogate all 256 possible base-pair combinations at a ligation junction in a single experiment. Rapid screening of ligase fidelity in a 96-well plate format has allowed the study of ligase fidelity in unprecedented depth. As an example of this new method, herein we report the ligation fidelity of Thermus thermophilus DNA ligase at a range of temperatures, buffer pH and monovalent cation strength. This screen allows the selection of reaction conditions that maximize fidelity without sacrificing activity, while generating a profile of specific mismatches that ligate detectably under each set of conditions.